Humanized and chimeric anti-factor bb antibodies and uses thereof

ABSTRACT

A method of inhibiting complement activation mediated by Bb inhibitors in a subject includes administering a Bb inhibitor to the subject to inhibit at least one of Bb binding to factors B and properdin, inhibit C3 cleavage, inhibit the activation of neutrophils, monocytes, platelets, and endothelium; or inhibit the formation of C3a, C5a, and MAC.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/619,858, filed Apr. 3, 2012, the subject matter of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to humanized and chimeric antibodies andantigen-binding fragments thereof with reduced effector functions andimmunogenicity. The present invention further relates to the use ofantibodies for factor B for inhibiting the activity of C3bBb or PC3bBbcomplexes, which will further inhibit the proteolytic activity of factorB in C3/C5 convertases. The humanized and chimeric monoclonal antibodiesof this invention selectively block the binding of factor Bb to thePC3bB complex without inhibiting classical pathway activation. Theseantibodies do not inhibit the interaction of C3b to factor B andtherefore have a unique function. Such antibodies are useful treatmentsfor disease indications where alternative complement pathway plays apathological role.

BACKGROUND OF THE INVENTION

The complement system is activated via three distinct pathways; theclassical pathway, the lectin pathway and the alternative complementpathway (AP). The classical pathway is activated via antigen-antibodycomplexes. The lectin pathway is a variation of the classical pathwayand the alternative pathway is activated by foreign material, artificialsurfaces, dead tissues, bacteria, dead yeast cells.

Activation of the classical pathway generates C3a, C4a, C5a and C5b-9molecules which activates a variety of cells in response to hostdefense. In pathological conditions, as a result of activation of thealternative pathway, anaphylatoxins C3a, C5a are formed which activatecells and C5b-9 molecules also known as the membrane attack complex(MAC) that damage tissues. Collectively these molecules mediateinflammation via cellular activation and release of inflammatorymediators. In addition to the role of C5b-9 as a lytic pore-formingcomplex, there is strong evidence that the depositing of sublytic MACmay play an important role in inflammation.

The classical complement pathway is important for host defense againstpathogens. The alternative complement pathway is activated inpathological inflammation. Elevated levels of C3a, C5a, and C5b-9 havebeen found associated with multiple acute and chronic diseaseconditions. Therefore, inhibition of disease-induced AP activation isimportant for clinical benefit in the diseases where complementactivation plays a role in disease pathology.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Theactivities included in the complement biochemical cascade present apotential threat to host tissue. An example includes the indiscriminaterelease of destructive enzymes possibly causing host cell lysis. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects.

In a disease condition where AP activation contributes to diseasepathology, elevated levels of C3a, C5a and C5b-9 molecules are found inserum, plasma, blood or other body fluids representative of the disease.Production and inhibition of each of these molecules via differentmechanisms is important for diseases. One possible mechanism forinhibiting the formation of the PC3bBb complex is via the use of ananti-Bb antibody. Thus blocking/inhibiting or preventing AP activationvia depleting Bb, neutralizing Bb, or inactivating Bb remains animportant therapeutic strategy.

The present invention relates to developed humanized and chimericantibody sequences that are novel and provide targeted binding to factorBb. The binding of such humanized/chimeric antibodies to factor Bbprevents activity of convertase. Such antibodies also prevent conversionof PC3bB into PC3bBb via binding the factor D cleavage site on factor Bof the PC3bB complex. These antibodies do not inhibit properdin bindingto C3b. Anti-factor Bb agents that bind Bb and prevent PC3bBb activityand prevent formation of new C3 convertase include, but are not limitedto, monoclonal and polyclonal antibodies, chimeric, humanized, fullyhuman, and nano-antibodies, Full length and fragments thereof, includingIgG, Fab, Fab′, F(ab′)₂, and IgGs. The antibodies of the presentinvention inhibit the formation of C3a, C5a, and C5b-9 which driveinflammation and also amplify the AP activation process.

Aptamers, small molecules, and SiRNA can also neutralize Bb binding tothe PC3bB complex and prevent production of AP induced production ofC3a, C5a, and C5b-9. As a result, cellular activation, inflammation andrelease of inflammatory mediators are also prevented. Because APactivation is linked to various acute and chronic human diseases, theblockade with anti-Bb agents will also block the inflammation processproviding clinical benefit to mammals treated with the anti-Bbmonoclonal antibodies.

Complement is one of several factors involved in pathogenesis and couldbe a significant pathological mechanism that offers an effective pointfor clinical control. The need for effective complement inhibitory drugsis signified by growing recognition of the importance ofcomplement-mediated tissue injury in a variety of disease states.Despite this, currently there is a complete absence of approved drugsfor human use that specifically target and inhibit complementactivation.

Factor B plays a key role in the amplification loop of the alternativepathway since it provides the catalytic subunit, Bb, for C3-convertase(PC3bBb). Antibodies that inhibit C3b binding to Ba have been developedbut none that inhibit the activity of the Bb. Factor B by itself is azymogen with no known catalytic activity. After binding to PC3b complex,factor B is cleaved by factor D to release Ba. It has been shown thatfactor B binds C3b through regions found within each of the Ba and Bbsubunits Inhibitors of factor Bb binders should results of selectiveinhibition of factor B function, thereby preventing formation of C3a,C5a and C5b-9, which are responsible for many deleterious effects.

Based on the results described in this patent application, we developedhumanized and chimeric antibodies that bind the catalytic domain offactor B and prevent the activity of the PC3bBb, bind the factor Dcleavage site on Bb and prevent the formation of additional PC3bBbmolecules. These antibodies do not inhibit C3b binding to factor B assuch binding events are shown to be mediated via Ba domain of factor B.This application developed humanized and chimeric anti-Bb specificinhibitors or inhibition methods that (a) will prevent factor B functionby blocking PC3bBb activity and/or (b) suppress factor B cleavage thatprevents Bb generation. These inhibitors appear to be inactivators ofthe C3 convertase enzymatic activity without disrupting the factor Binteraction with C3b. We have evaluated the inhibitory activity of theanti-factor Bb antibodies for their potential role in blocking the APactivation. These antibodies prevent factor B function both in vitro andin whole blood. Other anti-factor Ba monoclonal antibodies have alsobeen developed and tested in animal models of disease but not part ofthe current invention. These anti-Ba antibodies prevent factor B bindingto C3b and hence block the activation of the complement cascade.

This invention is designed to inhibit the functional activity of Bb andits progressive effects in pathological conditions by use of an anti-Bbantibody.

SUMMARY OF THE INVENTION

The present invention relates to a method of inhibiting Bb dependentcomplement activation by blocking Bb site on PC3bBb and C3bBb, andlimiting factor D mediated factor B cleavage. Antibodies that bind Bband inhibit only alternative pathway activation without inhibiting theclassical pathway are covered under this invention. Factor B dependentcomplement activation can be inhibited by a factor B inhibitor moleculeother than antibody such as aptamer and SiRNA. A factor B inhibitormolecule antibody can comprise a whole or fragmented anti-factor Bantibody. The fragmented anti-factor B antibody can be F_(ab),F_((ab)2), F_(v), or single chain F_(v). The anti-factor B antibody maybe monoclonal, polyclonal, chimeric, or de-immunized and have theability to bind factor B and its fragments. The present inventiondiscloses the use of Bb antibodies and not anti-Ba antibodies for thetreatment of several inflammatory disorders regulated via complementactivation.

In one aspect, the present invention relates to a method of inhibitingthe adverse effects of Bb-dependent complement activation in a subject.The method includes administering to the subject an amount of a Bbinhibitory agent effective to inhibit Bb-dependent complementactivation. In this context, the phrase “Bb-dependent complementactivation” refers to activation of all three complement pathways. Insome aspects of the invention, the Bb inhibitory agent is an anti-factorBb antibody or fragment thereof and, in other aspects, the anti-factorBb antibody has a reduced effector function. In still other aspects, theBb inhibitory agent is a Bb inhibitory peptide. The methods,compositions, and medicaments of the invention are useful for inhibitingthe adverse effects of Bb-dependent complement activation in vivo inmammalian subjects, including humans suffering from acute or chronicpathological conditions where inappropriate complement activation isinvolved in disease pathology.

In another aspect, the present invention creates an anti-factor Bbantibody containing various combinations of complementarity determiningregions 1 through 3 (CDRs 1 through 3), and framework regions (FR1through FR4). The CDRs include light and heavy chain combinations.CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 and the framework regionsare used for the treatment of the above mentioned disease conditions forwhich complement plays a role. Antibodies that contain the CDRs in anyorder within the amino acid sequence of the variable region are coveredunder this invention. As such, this invention covers the sequencesdiscussed as well as any sequence changes in the CDR or frameworkregions as long as 90% sequence identity is maintained. Such antibodiesbind only the Bb molecule with a stoichiometry ratio of 1:1, which meansthat by using the antibody, one can evaluate the percent activation in asample of plasma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the two complement pathways; the classical and thealternative complement pathways. This illustration separates the twopathways and not the convergence of the two pathways at C3b.

FIG. 2 illustrates the binding affinity of humanized anti-Bb antibody toB protein.

FIG. 3 illustrates the binding affinity of humanized anti-Bb antibody toBb protein.

FIG. 4 illustrates the humanized anti-Bb antibody does not inhibitFactor B binding to C3b.

FIG. 5 illustrates that the humanized anti-Bb inhibits the formation ofnew C3b molecules that form the new C3/C5 convertase

FIG. 6 illustrates that the humanized anti-Bb inhibits the formation ofnew PC3b complex.

FIG. 7 illustrates that the humanized anti-Bb inhibits the formation ofnew factor B/Bb molecules that are associated with C3/C5 convertase.

FIG. 8 illustrates that humanized anti-Bb inhibits the formation ofC5b-9 formation required for erythrocyte lysis and tissue injury.

FIG. 9 illustrates that humanized anti-Bb antibody inhibits thealternative pathway dependent lysis of erythrocytes.

FIG. 10 illustrates that humanized anti-Bb antibody does not inhibit theclassical pathway activation.

FIG. 11 illustrates that humanized antibody inhibits lysis of rabbiterythrocytes in PNH serum.

FIG. 12 illustrates the sequence of the murine anti-Bb antibody (SEQ IDNO:1 and SEQ ID NO: 2). Sequences of humanized antibody (SEQ ID NO: 3through SEQ ID NO:12) are presented.

FIG. 13 through FIG. 17 illustrates Sequence ID NO for CDRs andFramework regions.

FIG. 18 shows the peptide motif in Bb protein responsible for murine andhumanized anti-Bb binding.

DETAILED DESCRIPTION OF THE INVENTION

Standard terminologies including those used by skilled in the art arecommon and standard and have been used throughout the applicationwithout reservation.

The following definitions are provided in order to provide clarity withrespect to the terms as they are used in the specification and claims,in order to describe the present invention.

As used herein, the term “alternative pathway” refers to complementactivation, which has traditionally been thought to arise fromproteolytic generation of C3b from complement factor C3, for example, byzymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) fromGram-negative outer membranes, and rabbit erythrocytes, as well as frommany pure polysaccharides, rabbit erythrocytes, viruses, bacteria,animal tumor cells, parasites and damaged cells.

As used herein, the term “antibody” encompasses antibodies and antibodyfragments, which specifically bind to Bb or its polypeptides orportions, in which the antibody is derived from any antibody-producingmammal (e.g., a mouse, a rat, a rabbit, or a primate, including ahuman). Exemplary antibodies include polyclonal, monoclonal andrecombinant antibodies; multi-specific antibodies (e.g., bi-specificantibodies), humanized antibodies; murine antibodies, chimeric (i.e.mouse-human, mouse-primate, primate-human), monoclonal antibodies, andanti-idiotype antibodies, as well as de-immunized antibodies, and may beany intact molecule or fragment thereof.

As used herein, the term “antibody fragment” refers to a portion derivedfrom or related to a full-length anti-factor Bb antibody, generallyincluding the antigen binding or variable region thereof. Illustrativeexamples of antibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂ and Fvfragments, scFv fragments, diabodies, linear antibodies, single-chainantibody molecules and multispecific antibodies formed from antibodyfragments.

As used herein, the term “Bb inhibitory agent” refers to any agent thatbinds to or interacts with Bb and effectively inhibits Bb-dependentcomplement activation, including anti-Bb antibodies and Bb bindingfragments thereof, natural and synthetic peptides. Bb inhibitory agentsuseful in the method of the invention may reduce Bb-dependent complementactivation, therefore all activation, by greater than 20%. In oneembodiment, the Bb inhibitory agent reduces complement activation bygreater than 90%.

As used herein, a “chimeric antibody” is a recombinant protein thatcontains the variable domains and complementarily-determining regionsderived from a non-human species (e.g., rodent) antibody, while theremainder of the antibody molecule is derived from a human antibody.

As used herein, the term “classical pathway” refers to both (1)complement activation of the C1-complex triggered by an antibody boundto a foreign particle and requires binding of the recognition moleculeC1q, and also to (2) complement activation that occurs viaantigen-antibody complex formation.

As used herein, a “humanized antibody” is a chimeric antibody thatcomprises a minimal sequence conforming to specificcomplementarily-determining regions derived from non-humanimmunoglobulin that is transplanted into a human antibody framework.Humanized antibodies are typically recombinant proteins in which onlythe antibody complementarily-determining regions are of non-humanorigin.

As used herein, the “membrane attack complex” (“MAC”) refers to acomplex of the five terminal complement components (C₅-C₉) that insertsinto and disrupts membranes. MAC can also be referred to as C5b-9, andSC5b-9.

As used herein, “a subject” includes all mammals, including, but notlimited to, dogs, cats, horses, sheep, goats, cows, rabbits, pigs,humans, non-human primates, and rodents. The alternative pathway canalso provide an amplification loop for complement activation initiallytriggered via the classical and lectin pathways, in addition to itswidely accepted role as an independent pathway for complementactivation.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains, which enables the scFv to form the desired structure forantigen binding.

The antibodies of the present invention are chimeric and humanized antiBb monoclonal antibodies (mAb), and antigen binding fragments for thetreatment of human diseases. The antibody of the present invention willprovide high affinity antibodies useful to meet this need. The anti-Bbmonoclonal antibody of this invention neutralizes the catalytic activityof Bb in a PC3bBb complex and protects the Factor D cleavage site onFactor B in PC3bB complex.

Both animal and plant derived anti-Bb monoclonal antibodies withcharacteristic CDR(s) that binds and inhibits Bb binding to the PC3bBcomplex are covered under the present invention. CDRs having greaterthan 60% homology between CDRs of the antibody of the present inventionare covered under the present invention. A mouse monoclonal antibodyused to generate the chimeric and humanized anti-Bb antibody is coveredunder the present invention.

The antibody of the present invention differs from the prior art in thatthis antibody a) does not inhibit the binding of Factor B to C3b, b)binds the factor D cleavage site on factor B, c) has no affect on theclassical complement pathway, and d); binds Bb with 1:1 molarequivalence.

The humanized and murine anti-Bb antibodies inhibits the lysis ofrabbits erythrocytes in sera from normal and disease.

The humanized and murine anti-Bb antibodies inhibit the production ofC3a, C5a, C5b-9 and TNF alpha. These antibodies were generated againstBb and therefore do not cross react with Ba which is a fragment of theintact Ba molecule.

The antibodies of the present invention inhibit the formation of newC3b, PC3b, PC3bB, and PC3bBb. These antibodies exclusively inhibit thealternative pathway without having an effect on the amplification loopon the classical pathway.

Anti-factor Bb antibodies can be selected based on their ability toneutralize the Bb generated by the activation of the alternativepathway. The molar equivalence of 1:1 dictates that all of Bb isneutralized by the anti-Bb antibody.

Antibodies of the present invention have no effect on classical pathwayactivation. Thus, the monoclonal antibodies of the present invention donot inhibit the classical pathway. Inhibition of alternative pathway(amplification loop) should affect the classical pathway but theseantibodies do not show any effect on the classical pathway activationeven in situation where both pathways are generally activated.

Another aspect of the invention relates to antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of mAb, orcombinations thereof. The amino acid sequences of the variable heavychain CDR 1, 2 and 3 regions are shown in SEQ ID NO's: 14, 16, and 18,respectively. The amino acid sequences of the variable light chain CDR1, 2 and 3 regions are shown in SEQ ID NO's: 21, 23, and 25,respectively wherein the antibody specifically binds human Bb and B.

The antibody can be, for example, a chimeric antibody, humanizedantibody, human antibody, a humanized antibody or a chimeric antibodyThe CDRs within the variable region may range from 90% similar to about99% similar.

The antibodies of the present invention, the CDRs of the presentinvention, and the framework of the present invention are listed inFIGS. 12-17.

The antibodies of the present invention can be used to inhibitcomplement activation via the alternative pathway in vivo in subjects,including humans, suffering from an acute or chronic pathologicalinjury. The present invention can be used in conjunction with thefollowing diseases, disorders, injuries, and treatments, including butnot limited to:

Extracorporeal circulation diseases and disorders: Post-cardiopulmonarybypass inflammation, post-operative pulmonary dysfunction,cardiopulmonary bypass, hemodialysis, leukopheresis, plasmapheresis,plateletpheresis, heparin-induced extracorporeal LDL precipitation(HELP), postperfusion syndrome, extracorporeal membrane oxygenation(ECMO), cardiopulmonary bypass (CPB), post-perfusion syndrome, systemicinflammatory response, and multiple organ failure.

Cardiovascular diseases and disorders: acute coronary syndromes, Kawaskidisease (arteritis), Takayasu's arteritis, Henoch-Schonlein purpuranephritis, vascular leakage syndrome, percutaneous coronary intervention(PCI), myocardial infarction, ischemia-reperfusion injury followingacute myocardial infarction, atherosclerosis, vasculitis, immune complexvasculitis, vasculitis associated with rheumatoid arthritis (also calledmalignant rheumatoid arthritis), systemic lupus erythematosus-associatedvasculitis, sepsis, arteritis, aneurysm, cardiomyopathy, dilatedcardiomyopathy, cardiac surgery, peripheral vascular conditions,renovascular conditions, cardiovascular conditions, cerebrovascularconditions, mesenteric/enteric vascular conditions, diabetic angiopathy,venous gas embolus (VGE), Wegener's granulomatosis, heparin-inducedextracorporeal membrane oxygenation, and Behcet's syndrome.

Bone/Musculoskeletal diseases and disorders: arthritis, inflammatoryarthritis, non-inflammatory arthritis, rheumatoid arthritis, juvenilerheumatoid arthritis, systemic juvenile rheumatoid arthritis,osteoarthritis, osteoporosis, systemic lupus erythematosus (SLE),Behcet's syndrome, and Sjogren's syndrome.

Transplantation diseases and disorders: transplant rejection, xenograftrejection, graft versus host disease, xenotransplantation of organs orgrafts, allotransplantation of organs or grafts, and hyperacuterejection.

Eye/Ocular diseases and disorders: wet and dry age-related maculardegeneration (AMD), choroidal neurovascularization (CNV), retinaldamage, diabetic retinopathy, diabetic retinal microangiopathy,histoplasmosis of the eye, uveitis, diabetic macular edema, diabeticretinopathy, diabetic retinal microangiopathy, pathological myopia,central retinal vein occlusion (CRVO), corneal neovascularization,retinal neovascularization, retinal pigment epithelium (RPE),histoplasmosis of the eye, and Purtscher's retinopathy.

Hemolytic/Blood diseases and disorders: sepsis, systemic inflammatoryresponse syndrome” (SIRS), hemorrhagic shock, acute respiratory distresssyndrome (ARDS), catastrophic anti-phospholipid syndrome (CAPS), coldagglutinin disease (CAD), autoimmune thrombotic thrombocytopenic purpura(TTP), endotoxemia, hemolytic uremic syndrome (HUS), atypical hemolyticuremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH),sepsis, septic shock, sickle cell anemia, hemolytic anemia,hypereosinophilic syndrome, and anti-phospholipid syndrome (APLS).

Respiratory/Pulmonary diseases and disorders: asthma, Wegener'sgranulomatosis, transfusion-related acute lung injury (TRALI),antiglomerular basement membrane disease (Goodpasture's disease),eosinophilic pneumonia, hypersensitivity pneumonia, allergic bronchitisbronchiecstasis, reactive airway disease syndrome, respiratory syncytialvirus (RSV) infection, parainfluenza virus infection, rhinovirusinfection, adenovirus infection, allergic bronchopulmonary aspergillosis(ABPA), tuberculosis, parasitic lung disease, adult respiratory distresssyndrome, chronic obstructive pulmonary disease (COPD), sarcoidosis,emphysema, bronchitis, cystic fibrosis, interstitial lung disease, acuterespiratory distress syndrome (ARDS), transfusion-related acute lunginjury, ischemia/reperfusion acute lung injury, byssinosis,heparin-induced extracorporeal membrane oxygenation, anaphylactic shock,and asbestos-induced inflammation.

Central and Peripheral Nervous System/Neurological diseases anddisorders: multiple sclerosis (MS), myasthenia gravis (MG), myastheniagravis, multiple sclerosis, Guillain Bane syndrome, Miller-Fishersyndrome, stroke, reperfusion following stroke, Alzheimer's disease,multifocal motor neuropathy (MMN), demyelination, Huntington's disease,amyotrophic lateral sclerosis (ALS), Parkinson's disease, degenerativedisc disease (DDD), meningitis, cranial nerve damage from meningitis,variant Creutzfeldt-Jakob Disease (vCJD), idiopathic polyneuropathy,brain/cerebral trauma (including, but not limited to, hemorrhage,inflammation, and edema), and neuropathic pain.

Trauma-induced injuries and disorders: hemorrhagic shock, hypovolemicshock, spinal cord injury, neuronal injury, cerebral trauma, cerebralischemia reperfusion, crush injury, wound healing, severe burns, andfrostbite.

Renal diseases and disorders: renal reperfusion injury,poststreptococcal glomerulonephritis (PSGN), Goodpasture's disease,membranous nephritis, Berger's Disease/IgA nephropathy,mesangioproliferative glomerulonephritis, membranous glomerulonephritis,membranoproliferative glomerulonephritis (mesangiocapillaryglomerulonephritis), acute postinfectious glomerulonephritis,cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schonleinpurpura nephritis, and renal cortical necrosis (RCN).

Reperfusion injuries and disorders of organs: including but not limitedto heart, brain, kidney, and liver.

Reproduction and urogenital diseases and disorders: painful bladderdiseases and disorders, sensory bladder diseases and disorders,spontaneous abortion, male and female diseases from infertility,diseases from pregnancy, fetomaternal tolerance, pre-eclampsia,urogenital inflammatory diseases, diseases and disorders from placentaldysfunction, diseases and disorders from miscarriage, chronic abacterialcystitis, and interstitial cystitis.

Skin/Dermatologic diseases and disorders: burn injuries, psoriasis,atopic dermatitis (AD), eosinophilic spongiosis, urticaria, thermalinjuries, pemphigoid, epidermolysis bullosa acquisita, autoimmunebullous dermatoses, bullous pemphigoid, scleroderma, angioedema,hereditary angioneurotic edema (HAE), erythema multiforme, herpesgestationis, Sjogren's syndrome, dermatomyositis, and dermatitisherpetiformis.

Gastrointestinal diseases and disorders: Crohn's disease, CeliacDisease/gluten-sensitive enteropathy, Whipple's disease, intestinalischemia, inflammatory bowel disease, and ulcerative colitis.

Endocrine diseases and disorders: Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, stress anxiety, and other diseases affectingprolactin, growth or insulin-like growth factor, adrenocorticotropinrelease, pancreatitis, Addison's disease, diabetic conditions including,but not limited to, type 1 and type 2 diabetes, type I diabetesmellitus, sarcoidosis, diabetic retinal microangiopathy, non-obesediabetes (IDDM), angiopathy, neuropathy or retinopathy complications ofIDDM or Type-2 diabetes, and insulin resistance.

Treatment of Malignancies: diseases and disorders arising fromchemotherapeutics and radiation therapy.

The antibodies of the present invention can be therapeutic. Murine,chimeric, humanized, and primatized antibodies are currently consideredtherapeutic. However with recent advances in science, the antibody canalso be replaced by other types of antibodies in which the interactionof the antibody like molecule may fall within the range of low pMole tohigh pMole to low nMole.

Both the chimeric antibodies and the humanized antibodies have humanframework constant regions. The framework regions of the humanized andhuman are either natural human framework regions or the altered humanframework regions in order to increase the affinity and efficacy of thesaid CDR regions. Constant regions may or may not be present in the saidantibody. Various methods are available to produce antibodies with andwithout the constant regions in plants, bacterial and mammalian cellsystem.

Functional activity of the anti-Bb antibody is defined as the ability ofthe anti-Bb antibody to inhibit only AP activation without affecting theamplification loop of the classical pathway. These antibodies (1)inhibit to the catalytic activity of the PC3bB complex, (2) reducePC3bBb formation and/or C3bBb formation, (3) reduce concentration offree C3b, (4) reduce formation of C3b, (5) reduce formation of C3a, C5aand C5b-9, (6) reduces monocytes CD11b expression, (7) reducesneutrophil CD11b expression, (8) reduces platelet CD62 P expression, (9)reduces leukocyte-platelet conjugate formation, (10) reduces tumornecrosis factor alpha (TNF), and (11) reduces neutrophil elastaseformation.

Bispecific antibodies can be generated that can comprise (i) twoantibodies one with a specificity to Bb and another to a second moleculethat are conjugated together, (ii) a single antibody that has one chainspecific to Bb and a second chain specific to a second molecule, or(iii) a single chain antibody that has specificity to Bb and the othermolecule. Such bi-specific antibodies can be generated using techniquesthat are well known in the art.

The anti-Bb antibody or fragments thereof can be used in therapeuticmethods for the prophylactic and therapeutic treatment of diseasesmediated, directly or indirectly, by a component of the alternativecomplement pathway, and/or by a factor generated following activation ofthe alternative complement pathway.

The present invention also provides methods of inhibiting the adverseeffects of alternative pathway derived Bb-dependent complementactivation. The Bb inhibitory agents can be used alone as a primarytherapy or in combination with other methods as complement to enhancethe benefits of other treatments.

The inhibitory agents can be small molecules, aptamers, DNA fragments,small peptides representing CDR domains, SiRNA. These inhibitory agentsinhibit PC3bBb binding to C3, C5, C9, and C5b-9.

The Bb inhibitory agent can be administered in various ways byintra-arterial, intracranial, intravenous, subcutaneous, intramuscular,or other parenteral administration. Potentially orally fornon-peptidergic inhibitors, and most suitably by intra-arterial orintravenous administration. Administration may be repeated periodicallyas determined by a physician for optimal therapeutic effect.

EXAMPLES

Unless stated otherwise, all reagents were of high grade available. Allcomplement proteins, alternative and classical pathway buffers,detection antibodies, and erythrocytes were from Complement Technologies(Tyler, Tex.) or Quidel Corporation (San Diego, Calif.). Flow cytometryantibodies were from BD Biosciences, San Jose, Calif. TMB substrate wasfrom Kirkegaard & Perry Limited, Gaithersberg, Md. All secondaryantibodies were from American Qualex, San Clemente, Calif., BSA andother reagents were all from Sigma-Aldrich, St Louise, Mo.

ELISA plate readers (SpectraMax 190 and 250) were from MolecularDevices, and Flow Cytometer was FACS Calibur. Varity 3D program was usedfor data analyses, Curve fittings were done using MicroCal Originprogram. Hemolysis kinetic assay was run using SectraMax, MolecularDevices. ELISA plates were from Corning Costar, Lowell, Mass.

Humanized and chimeric antibodies contain the CDRs of the parent murinemonoclonal antibody, sequence (SEQ ID No 1 and SEQ ID No 2), which ispresent in this application. Mice were injected with human Bb(Complement Technology, Tyler, Tex.) and mouse serum was screened for Bbbinding and AP inhibitory activity. Spleen cells from properdin positivemouse were fused with myeloma cells using standard procedures. Thefusion cells were cloned into a single cell population using limitingdilution technique. The cells in 96 well plate were allowed to grow tosupernatant was tested using properdin binding and alternative pathwayinhibition. Cells that block AP activation were identified and furtherscreened using those that inhibit C5b-9 formation. These clones werecategorized under 1D3 which inhibit erythrocyte lysis. The antibodysecreting cell line was sequenced to generate an amino acid sequenceshown in SEQ ID NO: 1 and SEQ ID NO:2. The CDRs from both heavy andlight chains were grafted in the human framework to generate varioushumanized antibody sequences SEQ ID NO: 3 through SEQ ID NO: 12. Theantibody from SEQ ID NO 9 through 12 were produced in mammalian CHOcells, expressed, secreted and purified to produce a full IgG 1k. Theantibody was characterized as shown below.

Example 1 Humanized Anti-Bb Antibody Binds Factor B and Bb Proteins withHigh Affinity

The affinity of anti-Bb IgG1 and its antigen binding fragments to humanfactor B and Bb is in the range of low pM Antibody. Antibody binding toBb neutralizes the catalytic activity of the PC3bBb (C3/C5 convertase).Binding of anti-Bb to human factor B blocks the factor D cleavage siteon factor B. To perform these experiments, polystyrene microtiter plateswere coated with human factor B and Bb (2.0 μg/50 μl per well) inphosphate buffered saline (PBS) overnight at 4° C. After aspirating theproperdin solution, the wells were blocked with PBS containing 1% bovineserum albumin (BSA) (Sigma-Aldrich, St. Louis, Mo.) for 1 hour at roomtemperature. Wells without peptide or properdin coating served asbackground controls. Aliquots of humanized anti-factor Bb were added tothe B and Bb coated wells and allowed to incubate for 1 hour to allowbinding to occur. Following a 1 hour incubation period at roomtemperature, the plate was rinsed with PBS five times and incubated with1:2000 diluted detection peroxidase-conjugated goat anti-humanmonoclonal antibody. Following this incubation, the plate was rinsed andthe bound peroxidase was identified using TMB reagent.

As shown in FIGS. 2 and 3, humanized anti-Bb binds factor B (FIG. 2) andBb (FIG. 3) with picomolar affinity.

Example 2 Humanized Anti-Bb Monoclonal Antibody does not Inhibit FactorB Binding to C3b

Anti-factor B antibodies of previous inventions have typically inhibitedB interaction with C3b. This novel antibody does not inhibit the bindingof Factor B to C3b. In a typical experiment, polystyrene microtiterplate wells (96-well medium binding plates, Corning Costar, Cambridge,Mass.) were coated with C3b (2 μg/50 μl/well, complement technology,Tyler, Tx) in phosphate-buffered saline (PBS) pH 7.4 overnight at 4° C.After aspirating the C3b solution, wells are blocked with PBS containing1% bovine serum albumin (BSA; Sigma Chemical) for 1 h at roomtemperature. Wells without C3b coating serve as the background controls.Aliquots of humanized anti-Bb were added to solution containing thefixed concentration of Factor B. Following 1 h incubation at roomtemperature, the wells are extensively rinsed with PBS. The total bound“B” was detected with the polyclonal anti-B antibody. None of thehumanized anti-Bb concentrations inhibited the binding of factor B toC3b, as shown in FIG. 4.

Example 3 Humanized Anti-Factor Bb Inhibits the Formation of C3/C5Convertase (PC3bBb) of Alternative Complement Pathway

Alternative complement pathway is activated in normal human serum bylipopolysaccharide from Salmonella Typhosa. We have utilized thisparadigm to demonstrate whether anti-properdin antibody of thisinvention would inhibit the formation of PC3bBb. We measured thedeposition of P, C3b, Bb, and C5b-9 in the presence and absence thehumanized anti-Bb antibody. The deposited P, C3b, Bb, and C5b-9 weredetected with appropriate antibodies. In the presence of humanizedanti-Bb antibodies, a dose dependent inhibition of C3 and C5 convertaseformation was noticed as indicated by the inhibition of deposition ofeach of the P, C3b, Bb, and C5b-9 molecules.

In a typical assay, polystyrene microtiter plate wells were coated withLPS (Lipopolysaccharide from Salmonella Typhosa) at 2 μg/50 μl in PBSovernight. The wells were incubated with 1% BSA in PBS to block theunoccupied sites in the wells. Following a 2-hour blocking at roomtemperature and rinsing with PBS, normal human serum (10%) in an APbuffer was mixed with varying concentrations of the antibody andfragments. The mixture was incubated onto LPS coated wells. The platewas incubated for 2 hours at 37° C. to allow complement AP activation tooccur. Following incubation, the plates were extensively washed withPBS, and components of the C3 convertase were detected with theappropriate antibodies. We detected C3b with rabbit anti-human C3c at1:2000 in blocking solution, goat anti-human P at 1:2000 in blockingsolution and goat anti-human factor Bb at 1:500 in blocking solution andHRPO conjugated anti-human C5b-9 at 1:2000 in blocking solution. Plateswere incubated with their respective antibodies for 1-hour at roomtemperature. Following the incubation, the plates were rinsed with PBSand the bound antibodies were detected with peroxidase labeled goatanti-rabbit at 1:2000 for C3b and peroxidase labeled rabbit anti-goat at1:2000 in blocking solution for P detection. All plates were developedwith TMB following extensive washing with PBS. The blue color wasquenched with 1 M orthophosphoric acid. The presence of C3b, P and Bband MAC together are indicative of C3/C5 convertase formation.

FIG. 5 shows a dose dependent inhibition of C3b deposition, FIG. 6 showsa dose dependent deposition of P deposition, FIG. 7 shows a dosedependent deposition of Bb formation, and FIG. 8 shows a dose dependentdeposition of C5b-9 deposition by humanized anti-Bb antibodies. Thesedata provide direct evidence that anti-Bb monoclonal antibodies preventconvertase formation and inhibit AP activation.

Example 4 Humanized Anti-Bb Antibody Inhibits Alternative Pathway (AP)Dependent Lysis of Rabbit Red Blood Cell (rRBC)

This erythrocyte lysis assay is based on the formation of terminalcomplement complex on the surface of the rRBC. As a result, the rRBCsare lysed. The progressive decrease in light scatter at 700 nm is adirect measure of erythrocyte lysis. Typically, rRBC(s) are incubated innormal human serum in gelatin veronal buffer containing 5 mM MgCl₂.Under these conditions, the surface of rRBC triggers the activation ofalternative pathway in normal human serum. The alternative pathwayactivation leads to the formation of C5b-9 complex on the surface of therRBC(s). Agents that inhibit the formation of C5b-9 complexes areexpected to inhibit cellular lysis. To evaluate the effect ofanti-properdin antibody and fragments thereof, various concentrations ofIgG, F(ab′)₂, and Fab were incubated with normal human serum (10% NHS)in AP buffer at 37° C. with a fixed concentration of rabbit erythrocytesin a temperature controlled ELISA plate reader capable of reading at 700nm. A progressive decrease in light scatter (due to lysis of intactcells) was measured at 700 nm as a function of time. The data wererecorded and analyzed with a SpectraMax 190 plate reader and SoftMaxsoftware. For calculation total inhibition was calculated at eachconcentration of the IgG, F(ab′)2, and Fab, and the results wereexpressed as a % of unlysed controls. Data at each concentration wasplotted in a sigmoidal plot with MicroCal Origin Software.

As shown in FIG. 9, Humanized anti-Bb IgG inhibits AP dependenthemolysis of rRBC in normal human serum with an IC₅₀ of 79 nM in normalhuman serum inhibiting erythrocyte lysis. The antibodies are able toinhibit lysis with an IC₅₀ of approximately 79 nM.

Similar experiment was conducted with serum from Paroxysmal NocturnalHemoglobinuria (PNH) patients. Instead of 10% final serum concentration,we used 40% final serum concentration. As shown in FIG. 11, Humanizedanti-Bb prevented the lysis of erythrocytes in PNH serum.

Example 5 Humanized Anti-Bb does not Inhibit the Classical PathwayActivation

Monoclonal antibodies of the present invention do not inhibit theclassical pathway which is required for host defense. Antibodysensitized sheep erythrocytes were incubated with 10% normal human serumin gelatin veronal buffer containing calcium (5 mM CaCl₂/MgCl₂) buffer.Antibody sensitized sheep cells activate the classical pathway. As aresult, C5b-9 is formed on the surface of the erythrocyte acused lysis.We tested 10% normal human serum. Under both conditions, Anti-Bb did notinhibit sheep erythrocyte lysis. In a typical assay, 100 μl of antibodysensitized sheep erythrocytes (Complement Technologies, Tyler, Tex.)were incubated in 10% normal human serum in CP buffer to allowcomplement activation to occur. As a result of CP activation,erythrocytes undergo lysis. The progressive decrease in light scatteringdue to cellular lysis was measured at 700 nm as a function of time.

As shown in FIG. 10, humanized anti-Bb IgG does not inhibit the lysis ofthe antibody sensitized sheep cells normal human serum. These resultssuggest that anti-Bb antibodies are capable of selectively inhibitingthe alternative complement pathway without affecting the classicalpathway activation.

Example 6 Production of Humanized Anti-Bb Antibodies

Murine monoclonal antibody harboring the CDRs were sequenced and CDRswere grafted within various human framework regions. The antibody wascloned and expressed in CHO cells using methods developed in the art.FIGS. 12 through 17 show antibody CDR and framework regions.

Example 7 Epitope Mapping of the Bb Protein

The epitope mapping of the Bb protein was conducted by Pepscan usingclips technology. Multiple overlapping peptides were synthesized andantibody was allowed to bind the peptides at 1 μg/ml concentration.Peptides that produced the strongest signal were identified to be thepotential epitopes for the antibody. Sequences 47 & 48 were identifiedfor one antibody clone whereas sequences 49, 50, and 51 were identifiedfor the humanized anti-Bb antibody.

1-53. (canceled)
 54. An isolated anti-Bb antibody or antigen bindingportion thereof comprising a heavy chain variable domain including 3CDRs having at least 90% sequence identity to the amino acid sequencesof SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18 and a light chainvariable domain including 3CDRs having at least 90% sequence identity tothe amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO:25.
 55. The anti-Bb antibody or antigen binding portion thereof of claim54, comprising a heavy chain variable domain including 3 CDRs havingamino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18and a light chain variable domain including 3CDRs having of SEQ ID NO:21, SEQ ID NO: 23, and SEQ ID NO:
 25. 56. The anti-Bb antibody orantigen binding portion thereof of claim 54, comprising humanized framework regions, wherein the humanized framework regions are selected fromSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:12.
 57. The anti-Bb antibody or antigen binding portion thereof of claim54, comprising humanized or non-natural framework regions, wherein thehumanized or non-natural framework regions are selected from SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, and SEQ ID NO:
 26. 58. The anti-C3b antibody orantigen binding portion thereof of claim 54, comprising humanized ornon-natural framework regions, wherein the humanized or non-naturalframework regions are selected from SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 30, andSEQ ID NO:
 26. 59. The anti-Bb antibody or antigen binding portionthereof of claim 54, comprising humanized or non-natural frameworkregions, wherein the humanized or non-natural framework regions areselected from SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO:
 26. 60.The anti-Bb antibody or antigen binding portion thereof of claim 54,comprising humanized or non-natural framework regions, wherein thehumanized or non-natural framework regions are selected from SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 19, SEQ ID NO: 46, SEQ IDNO: 40, SEQ ID NO: 41, and SEQ ID NO:
 42. 61. The anti-Bb antibody orantigen binding portion thereof of claim 54, wherein the anti-Bbantibody or antigen binding portion thereof binds to peptide regionslocated on factor Bb having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, and SEQ ID NO:
 51. 62. The anti-Bb antibody or antigen bindingportion thereof of claim 54, wherein the antibody or antigen bindingportion thereof antibody inhibits the formation of C3b via theinhibition of the formation of the PC3bBb complex.
 63. The anti-Bbantibody or antigen binding portion thereof of claim 54, wherein theantibody or antigen binding portion thereof inhibits the formation ofnewly produced C3a, C5a, SC5b-9.
 64. The anti-Bb antibody or antigenbinding portion thereof of claim 54, wherein the antibody or antigenbinding portion thereof inhibits the activation of neutrophil,monocytes, and platelets.
 65. The anti-Bb antibody or antigen bindingportion thereof of claim 54, wherein the antibody or antigen bindingportion thereof, inhibits the lysis of erythrocytes.
 66. Apharmaceutical composition comprising a therapeutically effective amountof an anti-Bb antibody or antigen binding portion thereof of claim 54.67. A method of treating a disorder wherein complement activationcontributes to the disorder pathology in a subject in need thereof,comprising: administering a therapeutically effective amount of ananti-Bb or antigen binding portion thereof that includes a heavy chainvariable domain including 3 CDRs having at least 90% sequence identityto the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ IDNO: 18 and a light chain variable domain including 3CDRs having at least90% sequence identity to the amino acid sequences of SEQ ID NO: 21, SEQID NO: 23, and SEQ ID NO:
 25. 68. The method of claim 67, wherein thedisorder is an inflammatory disorder or an autoimmune disease.
 69. Themethod of claim 67, wherein the disorder is an ocular disorder.
 70. Themethod of claim 69, wherein the ocular disorder is selected from thegroup consisting of wet and dry age related macular degeneration,choroidal neovascularization, uveitis, diabetic retinopathy, diabeticmacular edema, pathological myopia, Von Hippel-Lindau disease, diabeticretinopathy, histoplasmosis of the eye, diabetic retinopathy, choroidalneo-vascularization (CNV), Central Retinal Vein Occlusion (CRVO),corneal neo-vascularization, geographic atrophy, drusen disease, andretinal neovascularization.
 71. The method of claim 67, wherein thedisorder is selected from the group consisting of asthma, chronicobstructive pulmonary disease (“COPD”), allergic broncho-pulmonaryaspergillosis, hypersensitivity pneumonia, eosinophilic pneumonia,emphysema, bronchitis, allergic bronchitis bronchiecstasis, cycticfibrosis, tuberculosis, hypersensitivity pneumonitis, occupationalasthma, sarcoid, reactive airway disease syndrome, interstitial lungdisease, hyper-eosinophilic syndrome, rhinitis, sinusitis,exercise-induced asthma, pollution-induced asthma, cough variant asthma,parasitic lung disease, respiratory syncytial virus (“RSV”) infection,parainfluenza virus (“PIV”) infection, rhinovirus (“RV”) infection, andadenovirus infection.